Fig. 2

Single-cell RNA sequencing reveals different cell states after exposure to heterogeneous flow. a Workflow showing orbital flow setup used on PCV and normal BOEC lines prior to dissociation for scRNA sequencing. b CDH5 (green) and CAV1 (red) immunostainings reveal patterns of heterogeneous flow in the setup. Nuclei were stained with DAPI (blue). Right panel shows measurements of CAV1 intracellular re-distribution as an indicator of flow response (BOECs from 4 PCV and 3 normal individuals). Data shown are normalized to individual static conditions with median and quartiles indicated. p values were obtained using two-way ANOVA with Tukey’s multiple comparisons test. ****p < 0.0001. Scale bar, 100 μm. c Single-cell UMAP showing sequenced cells for all subjects (n = 19,288 cells, BOECs from 2 PCV and 2 normal individuals). We identified 5 distinct transcriptomic cell states across the integrated dataset, (1) Proinflammatory, (2) Migratory-Angiogenic, (3) Transitory-Proliferative, (4) Proliferative, and (5) Quiescent cells. d Gene expression of top 10 positive markers genes for each of the cell states shown in c. e Gene set enrichment analysis against GO (Biological processes) database of marker genes for two largest clusters shown here ranked by Bonferroni corrected p values